The invention relates to novel chemoattractants.
Six different types of white blood cells (leukocytes) are typically found in the blood. These are neutrophils, eosinophils, basophils, monocytes, lymphocytes, and plasma cells. The neutrophils and monocytes are primarily responsible for attacking and destroying invading bacteria, viruses, and other harmful agents. Neutrophils circulate within the bloodstream as mature, functional cells. Monocytes, however, circulate as immature cells that have a limited ability to fight infectious agents. It is only when monocytes are stimulated by chemotactic agents to move through the capillary wall into surrounding tissue that they become fully active. Once monocytes enter the tissues they begin to swell and many lysosomes and mitochondria appear in their cytoplasm. At this point, monocytes become called macrophages, which are extremely effective phagocytes. Each macrophage can engulf as many as 100 bacterial cells, as well as large particles, including whole red blood cells, malarial parasites, and necrotic tissue.
Chemokines are chemotactic cytokines that contribute to various immune and inflammatory responses by regulating the movement of selected blood-borne leukocytes into the tissues (Baggiolini et al., Adv. Immunol. 55:97-179, 1994; Oppenheim et al., Ann. Rev. Immunol. 9:617-648, 1991). All known chemokines have been assigned to one of three families on the basis of the chromosomal location of the genes that encode them and on the motif formed by conserved cysteine residues in the mature proteins. Two of these families, designated xcex1 and xcex2, have many members, all of which have four conserved cysteine residues. The first two cysteines of xcex1 chemokines are separated by a single amino acid (CXC motif), and these proteins are encoded by genes clustered on human chromosome 4. In contrast, the first two cysteines of all xcex2 family chemokines are adjacent to one another (CC motif), and these proteins are encoded by genes clustered on human chromosome 17.
Chemokines having the CXC motif (xcex1 chemokines) primarily affect neutrophils and lymphocytes, and they can modulate angiogenesis. Chemokines having the CC motif (xcex2 chemokines) affect monocytes, lymphocytes, eosinophils, and basophils with variable selectivity. In addition, certain chemokines in the xcex1 and xcex2 family can inhibit HIV replication in T cells and monocytes, respectively.
The third chemokine family currently has only one member: the T cell-specific chemoattractant, lymphotactin (Kelner et al., Science 266:1395-1399, 1994). The gene encoding lymphotactin is located on human chromosome 1, and the protein sequence contains only the second and fourth cysteines found in the xcex1 and xcex2 chemokine families (Kennedy et al., J. Immunol. 155:203-209, 1995).
The monocyte chemoattractant proteins (MCPs), which constitute a subfamily of the xcex2 chemokines described above, include the three human MCP proteins (MCP-1, MCP-2, and MCP-3). These proteins are xcx9c65% identical, and all have Gln as their N-terminal amino acid (Van Damme et al., J. Exp. Med. 176:59-65, 1992; Yoshimura et al., J. Exp. Med. 169:1449-1459, 1989).
The chemokine eotaxin is also a xcex2 chemokine. It is related in sequence to the MCP proteins, but does not contain an amino-terminal Gln residue. Eotaxin appears to be unique among the chemokines in that it causes the selective infiltration of eosinophils when injected subcutaneously and when administered directly to the lungs of naive guinea pigs.
Five genes encoding human xcex2 chemokine receptors, referred to as CKR1 through CKR5, have been cloned. (Charo et al., Proc. Natl. Acad. Sci. USA 91:2752-2756, 1994; Combadiere et al., DNA and Cell Biol. 14:673-380, 1995; Combadiere et al., J. Leukocyte Biol. in press, 1996; Gao et al., J. Exp. Med. 177:1421-1427, 1993; Neote et al., Cell 72:415-425, 1993; Power et al., J. Biol. Chem. 270:19495-19500, 1995; Samson et al., Biochem. 35:3362-3367, 1996). CKR2a and CKR2b are splice variants of the same gene. These receptors are not ubiquitously expressed: CKR1, CKR4, and CKR5 appear to be widely expressed on leukocytes, while expression of CKR2 and CKR3 is mainly restricted to monocytes and eosinophils, respectively.
The invention features substantially pure nucleic acid molecules that encode MCP-4 and MCP-5, two novel members of the xcex2-chemokine family. The invention also includes polypeptides encoded by these nucleic acid molecules.
The nucleic acid molecules may consist of genomic DNA, cDNA, or mRNA, and, due to the degenerate nature of the genetic code, may vary in sequence provided that the encoded polypeptides are MCP-4 or MCP-5, as shown, for example, in FIGS. 1A and 1B, respectively.
Substantially pure MCP-4 and MCP-5 polypeptides, biologically active fragments of these polypeptides, including immunogenic fragments, are also considered within the scope of the invention. The biological activity of any given fragment of MCP-4 or MCP-5 may be readily determined by conducting an assay of, e.g., monocyte chemotaxis, as described herein. The preferred polypeptides of the invention are both substantially homologous to MCP-4 or MCP-5 and retain the biological activity of the relevant polypeptide, as described herein.
The nucleic acid molecules can encode a mammalian MCP-4 or MCP-5 polypeptide, such as those from a human, mouse, rat, guinea pig, cow, sheep, horse, pig, rabbit, monkey, dog, or cat. Preferably, the nucleic acid molecule encoding MCP-4 encodes human MCP-4 and the nucleic acid molecule encoding MCP-5 encodes murine MCP-5 or, more preferably, human MCP-5.
The nucleic acid molecules can be placed under the control of a promoter, which may be constitutively active or induced by one or more external agents. The promoter can provide the means to achieve tissue-specific or cell type-specific expression of the nucleic acid molecules of the invention. Alternatively, or in addition, the nucleic acid molecules can be operably linked to a DNA regulatory sequence. Skilled artisans will recognize that the nucleic acid molecules can be placed into a vector construct, such as a plasmid or viral vector, which may in turn be used to transduce living cells with the nucleic acid molecules of the invention. Cells can be transfected with plasmid vectors by standard methods including, but not limited to, liposome-, polybrene-, or DEAE dextran-mediated transfection (see, e.g., Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413, 1987; Ono et al., Neurosci. Lett. 117:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989), electroporation (Neumann et al., EMBO J. 7:841, 1980), calcium phosphate precipitation (Graham et al., Virology 52:456, 1973; Wigler et al., Cell 14:725, 1978; Felgner et al., supra) microinjection (Wolff et al., Science 247:1465, 1990), or velocity driven microprojectiles (xe2x80x9cbiolisticsxe2x80x9d). Viruses known to be useful for gene transfer include adenoviruses, adeno associated virus, herpes virus, mumps virus, poliovirus, retroviruses, Sindbis virus, and vaccinia virus such as canary pox virus.
The nucleic acid constructs described above are useful in a variety of ways. For example, the constructs may be used as a source of recombinantly produced MCP-4 or MCP-5 polypeptides. Alternatively, the constructs may be administered themselves in a therapeutic approach, as described below. In addition, the constructs may be used to generate transgenic animals that overexpress, or fail to express, MCP-4 or MCP-5. The animal can be a mouse, a worm, or any other animal considered useful for research or drug development. Transgenesis has become routine in the art of molecular biology.
MCP-4 or MCP-5 polypeptides expressed by transfected cells can be purified and injected into an animal, such as a rabbit, in order to generate polyclonal antibodies that specifically bind MCP-4 or MCP-5. Monoclonal antibodies may also be prepared using standard hybridoma technology (see, e.g., Kohler et al., Nature 256:495, 1975; Kohler et al., Eur. J. Immunol. 6:292 and 6:511, 1976; Hammerling et al., In Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, 1981). Alternatively, in various embodiments, an immunologically-active antibody fragment, such as an Fabxe2x80x2, (Fabxe2x80x2)2, or genetically engineered Fv fragment (see U.S. Pat. No. 4,946,778, hereby incorporated by reference) may also be useful. Once generated, the antibodies may be purified according to standard methods and injected into a mammal, such as a human, in order to inhibit the inflammatory response caused by chemotaxis of various leukocytes, particularly monocytes.
Preferably, the nucleic acid molecules of the invention, cells that express these molecules, or a therapeutic composition containing the encoded polypeptides, are administered to a mammal in order to stimulate a local immune response. This approach would be particularly beneficial to mammals that are suffering from cancer. The cancer may be evident as, for example, a lymphoma (such as Hodgkin""s lymphoma), a plasmacytoma, a melanoma, a sarcoma, or a tumor within the lung or gastrointestinal tract.
In the event that the desired result is inhibition (rather than stimulation) of a local immune response, a mutant MCP-4 or MCP-5 polypeptide, antibodies or fragments thereof that specifically bind MCP-4 or MCP-5, or antisense MCP-4 or MCP-5 oligonucleotides may be administered. The mutant MCP-4 or MCP-5 polypeptides may lack, for example, between one and ten of the amino acid residues present at the amino terminus of the mature polypeptides. Alternatively, the mutant polypeptides may contain additional amino acid residues, for example between 3 and 10 amino acid residues, at the amino-terminus of the mature polypeptides. Where amino acid residues are added, they may be random or they may be selected to have particular biological properties such as stability or hydrophilicity. Antisense MCP-4 or MCP-5 oligonucleotides preferably consist of at least eight nucleotides, and have a sequence that is the reverse and complement of that found within the genes encoding these polypeptides.
Suppression or inhibition of the local immune response would be beneficial when, for example, the mammal has been diagnosed as having asthma, chronic obstructive pulmonary disease, cystic fibrosis, sinusitis, rhinitis, atherosclerosis, glomerulonephritis, multiple sclerosis, or an inflammatory bowel disease.
The nucleic acid molecules of the invention are also useful in treating a mammal that is suffering from an infectious disease, such as acquired immune deficiency syndrome (AIDS) or malaria. Preferably, the method of treatment consists of administering to the infected mammal an MCP-4 or MCP-5 polypeptide, or a fragment thereof that is sufficient to bind the chemokine receptors by which these infectious agents gain entry to the cell.
The local immune response may also be modulated by administering a compound that modulates the chemotactic activity of MCP-4 or MCP-5. Examples of such modulators include interferon-gamma, tumor necrosis factor-xcex1, interleukin-1, and interleukin-4. Additional modulatory compounds may be discovered by, for example, the method described herein, whereby a population of cells that express MCP-4 or MCP-5 is obtained, a subset of these cells is contacted with a candidate modulatory compound and the level of MCP-4 or MCP-5 expression in the subset of cells is compared with the level of expression in an equivalent number of cells in the initial population that were not contacted with the potential modulator. A difference in the level of MCP-4 or MCP-5 expression would indicate the presence of a compound that modulates MCP-4 or MCP-5 expression. Either gene expression or protein expression may be readily assessed by methods well known to skilled artisans. For example, mRNA expression could be assessed by Northern blot or RNAse protection analyses and protein expression could be assessed by Western blot analysis.
By xe2x80x9cpolypeptidexe2x80x9d is meant any chain of more than two amino acids, regardless of post-translational modification such as glycosylation or phosphorylation.
By xe2x80x9csubstantially purexe2x80x9d is meant that the substance in question, be it a nucleic acid molecule, a polypeptide, or an antibody, has been separated from the components that naturally accompany it. Typically, a polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, a preparation of MCP-4 or MCP-5 is at least 75%, more preferably at least 90%, and most preferably at least 99% pure, by weight. A substantially pure polypeptide may be obtained, for example, by extraction from a natural source (e.g., a mammalian cell), by expression of a recombinant nucleic acid encoding the polypeptide, or by chemically synthesizing the polypeptide. Purity can be measured by any appropriate method, such as column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis. In reference to a DNA molecule, xe2x80x9csubstantially purexe2x80x9d means that the molecule is free of the genes that flank it in the naturally-occurring genome of the organism from which it is obtained. The term therefore includes, for example, a recombinant DNA molecule which is incorporated into a vector; into an autonomously replicating plasmid or virus; into the genomic DNA or a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by the polymerase chain reaction (PCR) or by restriction endonuclease digestion). It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
By xe2x80x9csubstantially identicalxe2x80x9d to MCP-4 and MCP-5 is meant a sequence having at least 80% identity to the MCP-4 and MCP-5 sequences shown in FIGS. 1A and 1B. Preferably, the identity is 90% and most preferably the identity is 95%.
By xe2x80x9coperably linkedxe2x80x9d is meant that a gene or portion of a gene is connected to one or more regulatory elements, which are also a particular DNA sequences, in such a way as to permit expression of the gene when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory element(s).
The term xe2x80x9ctransgenicxe2x80x9d as used in reference to an animal or a cell, means an animal or cell into which a DNA sequence has been inserted by artifice and which becomes a part of the genome of the animal or cell.
By xe2x80x9cbiological activityxe2x80x9d of MCP-4 or MCP-5 is meant the biological activity (preferably monocyte chemoattractant activity) which is at least 50% of that observed using MCP-4 or MCP-5, having the sequence shown in FIGS. 1A and 1B respectively, in any of the assays describes herein. Preferably the activity is 80% of the activity observed with the MCP-4 and MCP-5 polypeptides of FIGS. 1A and 1B, and most preferably, the activity is 95% of that observed with the MCP-4 and MCP-5 polypeptides of FIGS. 1A and 1B.
By xe2x80x9cmodulatory compoundxe2x80x9d or xe2x80x9cmodulatorxe2x80x9d is meant any compound that is capable of increasing or decreasing the expression or activity of MCP-4 or MCP-5. Expression may be modified at the level of transcription, translation, or post-translation. Activity may be modified at the level of receptor binding or within the cytoplasm of a cell. Activity may be assessed as, for example, monocyte chemotaxis per unit of MCP-4 or MCP-5 protein. Activity would be considered stimulated if, for example, the number of monocytes in a target tissue is at least 20% greater than the number of monocytes in a control, untreated tissue that is in all other substantive ways identical to the treated tissue. Conversely, activity would be considered inhibited if, for example, the number of monocytes in a target tissue is at least 20% less than the number of monocytes in a control, untreated tissue that is in all other substantive ways identical to the treated tissue.
Other features and advantages of the invention will be apparent from the following description and from the claims.